I propose to test experimentally several hypotheses concerning mutation. I will construct tester DNA sequences by vitro manipulation of the bacteriophage T4 rIIB gene fragment cloned into M13. I will then insert the tester sequences into the rIIB gene and into an E. coli lac I-Z fusion gene that under the appropriate circumstances can express Beta-galactosidase. The tester sequences confer the null phenotype on the gene into which they are inserted. The tester sequences will be designed so that mutation along a specific pathway to be analyzed can be assayed genetically as "reversion" from rIIB- to rIIB+ or lac- to lac+. The tester sequences will be replicated by two different replication apparatuses--T4 and E. Coli. Thus I will be able to ascertain organism-determined specificity of mutation. The specific hypotheses that I plan to test are the dependence of frameshift mutation on the number of iterated bases at a site (the Streisinger hypothesis), and the role of identical or nearly identical directly repeated sequences in deletion formation and site mutation. It seems reasonable that the ways in which "real" DNA differs from random sequence DNA of the same nucleotide composition reflect mutational trends throughout evolution. I will test whether the dinucleotide disparities noted by Stormo reflect spontaneous mutation frequencies. In addition, I will ask if real DNA differs from random sequence DNA in other ways. The approach taken in this research utilizes molecular biology, genetics, in vitro recombinant DNA technology, chemical synthesis of DNA, and computer analysis of DNA sequences. Information about the intrinsic and organism-specific mutability of DNA will be useful for the field of genetic engineering--a field in which genetic stability is a desideratum.